In recent years, a certain treatment style has been widely spread, that is, a treatment style which involves treating a cell-containing fluid typically such as peripheral blood, bone marrow, umbilical cord blood, or tissue extracts, to isolate only cell fractions which are effective in the treatment, and then administering the cell fractions to a patient to enhance therapeutic effects, whereas unnecessary components are removed without administration, thereby reducing side effects more efficiently. An example is a hematopoietic stem cell transplant for the treatment of leukemia or solid cancers. During the transplant, only cells effective in the treatment (nucleated cells, including hematopoietic stem cells), which are separated from bone marrow or peripheral blood by removing red blood cells for purification, are administered to patients. Umbilical cord blood banking requires cells to be cryopreserved before use. Thus, it also requires separation and purification of nucleated cells to prevent red blood cell hemolysis, which may occur during cryopreservation. Further, transplantation of mononuclear cell fractions of bone marrow, umbilical cord blood, or peripheral blood has been used in clinical practice for the treatment of ischemic diseases, such as cerebral infarction, myocardial infarction, and limb ischemia. Such mononuclear cell fractions are rich in stem cells, typically such as mesenchymal stem cells, hematopoietic stem cells, and endothelial progenitor cells, and it is believed that the stem cells promote angiogenesis or nerve regeneration, thereby producing a therapeutic effect. Meanwhile, it is believed that granulocytes can cause side effects, such as inflammation, to reduce the therapeutic effect. Therefore, only cells which are effective in the treatment (a group of mononuclear cells, including stem cells) are separated from bone marrow or peripheral blood by removing granulocytes for purification, and administered to a patient.
For example, Taguchi et al. demonstrate that transplantation of bone marrow mononuclear cells containing CD34 positive cells to patients who suffer Buerger's disease (one of peripheral arterial occlusive diseases) accelarates angiogenesis and therefore can be an effective way to treat Buerger's disease (See Non Patent Literature 1). Nakano-Doi et al. have also reported that transplantation of bone marrow-derived mononuclear cells is effective as the treatment for patients who suffer acute ischemic diseases, typically such as cerebral infarction, or ischemic heart diseases (See Non Patent Literature 2).
Typical examples of a method for separation and purification of such nucleated cells or mononuclear cells include centrifugation, and density-gradient centrifugation using an isolation medium, typically such as the Ficoll solution. The density-gradient centrifugation method, however, has some disadvantages. For example, the method has a large physical impact on cells, and needs complicated operations and operations in an open system. Thus, this method requires a large facility, called cell processing center (CPC), to perform cell therapy.
Based on the above situation, as a cell separation method allowing simple operation in a closed system, a nucleated cell separation method using a device including non-woven fabrics is disclosed (see Patent Literature 1). Also, the following methods are disclosed: a method of removing leukocytes using a device in which non-woven fabrics that are processed by forming pores with a specific pore-area ratio to suppress clogging are laminated between two different filters (see Patent Literature 2); a method of efficiently recovering mononuclear cells using a cell separation filter that selectively captures granulocytes (see Patent Literature 3). However, these methods require non-woven fabrics, which originally lack versatility, to be designed to suit target cells in order to improve a recovery yield or reduce unnecessary cell contamination.
Considering the above disadvantages such as impacts on cells due to centrifugation, requirement for complicated operations, and the risk of contamination, a separation method using a cell separation filter which has a structure in a laminate of non-woven fabrics is preferred. Unfortunately, to improve performance of non-woven fabrics, the cell separation filter becomes poor in versatility, and it is only suitable for a specific cell. Thus, non-woven fabrics need to be developed to suit respective cell species, which will be disadvantageous. The solution for such problems has not yet been found so far.